Inherited mutations in SLC12A1 and SLC12A2, the genes encoding the renal electroneutral Na-Cl and Na-K-2Cl co-transporters, result in salt wasting disorders associated with changes in blood pressure. The syndromes, called Gittleman and Bartter, are due to decreased Na+-re-absorption in distal convoluted tubule and thick ascending limb of Henle, respectively. Two Sterile20 kinase, SPAK, and OSR1, are involved in regulating the co-transporters. The kinase directly binds to the N-terminal tails of the co-transporter and phosphorylates them. A whole-genome study found an association between SPAK and increased blood pressure, and SPAK knockout mice exhibit a Gittleman-like phenotype. Surprisingly, NKCC2 phosphorylation is increased in SPAK knockout animals, but is blunted in phosphorylation-deficient knock-in SPAK mice, suggesting a possible interaction between SPAK and OSR1 in the thick ascending limb. The purpose of this 2 PD/PI application is to understand the molecular details leading to increased Na-K-2Cl co-transporter phosphorylation in the SPAK knockout and to define the role of each kinase in modulating Na+-re-absorption in the two tubule segments. In this application, we propose to 1) examine the mechanisms of SPAK/OSR1 activation, determine the role of dimerization, and establish the existence of SPAK-OSR1 interactions; 2) examine the function of putative inhibitory isoforms and of Cab39, a scaffold calcium binding protein; and 4) establish the role of SPAK and OSR1 in regulating renal salt transport and maintaining blood pressure. The first two aims will involve functional studies using heterologous expression in both Xenopus laevis oocytes and mammalian kidney cells, as well as molecular studies involving protein-protein interaction both in vitro and in vivo. The third aim will consist of detailed phenotypic analysis of genetically-modified mice under different diet regiments, and the development and study of a novel constitutively active OSR1 knock-in mouse. This application is a logical extension of extensive studies of cation-chloride co-transporters and their regulation by the submitting PI and a natural extension of a successful ongoing AARA-funded Challenge grant collaboration between the two PD/PIs. Upon completion, the proposed studies will clarify the role of two kinase and demonstrate that they are integrated into precise signaling networks in the thick ascending limb of Henle and the distal convoluted tubule.